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Nanobodies raised against monomeric a-synuclein inhibit fibril-formation and destabilize toxic oligomeric species of a-synuclein


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Nanobodies raised against monomeric


-synuclein inhibit fibril formation and

destabilize toxic oligomeric species

Marija Iljina


, Liu Hong


, Mathew H. Horrocks


, Marthe H. Ludtmann


, Minee L. Choi


, Craig D. Hughes



Francesco S. Ruggeri


, Tim Guilliams


, Alexander K. Buell


, Ji-Eun Lee


, Sonia Gandhi


, Steven F. Lee



Clare E. Bryant


, Michele Vendruscolo


, Tuomas P. J. Knowles


, Christopher M. Dobson


, Erwin De Genst


and David Klenerman



Background:The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS.

Results:We show that both nanobodies inhibit the formation ofɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers ofɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity.

Conclusions:The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.

Keywords:Protein aggregation, Amyloid toxicity , Neurodegeneration, Aggregation inhibitors, Antibody, Single-molecule fluorescence


The aberrant aggregation of the proteinɑ-synuclein (ɑS) has been linked to the onset and progression of Parkin-son’s disease (PD), dementia with Lewy bodies [1], PD dementia, multiple system atrophy, and related synu-cleopathies [2–4]. The histopathological characteristics of PD and its associated disorders include the presence of neuronal inclusions, for example, Lewy bodies and Lewy neurites, which are primarily composed of fibrillar ɑS [1, 5]. The deposition of ɑS in the nervous system follows a characteristic pattern [6], and the ability of

aggregated species to propagate across the brain by a mechanism that is defined as prion-like is increasingly recognized [7]. In aqueous solution, monomeric ɑS self-assembles into amyloid fibrils resembling those that are deposited in the brain, and the aggregation process proceeds through the formation of intermediate species, including soluble oligomers, prior to fibril forma-tion [8, 9]. Moreover, these oligomeric species, rather than mature amyloid fibrils, have increasingly been identified as the most highly neurotoxic forms ofɑS [10–16].

As a consequence of the central role ofɑS aggregation in PD and the neurotoxicity of its assemblies, immuno-therapy againstɑS is being widely pursued as a potential disease-modifying strategy [17, 18]. Although passive immunization using antibodies targeting ɑS has shown promise in numerous in vitro and in vivo model systems,

* Correspondence:cmd44@cam.ac.uk;erwin.degenst@astrazeneca.com;

dk10012@cam.ac.uk †Equal contributors

1Department of Chemistry, University of Cambridge, Lensfield Road,

Cambridge CB2 1EW, UK

Full list of author information is available at the end of the article


as well as in several clinical trials [19, 20], there is no ef-fective treatment for PD and other synucleinopathies. In order to make significant advances in this objective, it is crucial to develop a detailed understanding of the mo-lecular mechanism of action of ɑS-specific antibodies during the aggregation process of the protein.

Over the years, full-length antibodies and antibody fragments, such as scFvs, have been generated against different regions and different species of ɑS for either biophysical studies, target validation, or therapy (reviewed in De Genst et al. [21] and Bergstrom et al. [20]), and have been used in multiple reported studies [22–27].

Full-length ɑS consists of 140 amino acid residues di-vided into three distinct regions, namely a positively charged lipid-binding N-terminal segment comprising residues 1–60, a central hydrophobic segment consisting of residues 61–95, and a negatively-charged C-terminal region comprising residues 96–140 [28]. This latter re-gion does not include the residues observed to be in the amyloid fibril core [29], although previous in vitro stud-ies have shown that its deletion promotesɑS aggregation [30], indicating its involvement in the aggregation process. Notably, antibodies directed to the C-terminal region ofɑS, or specific for oligomeric forms ofɑS, were reported both to suppress strongly its aggregation and to reduce cellular toxicity [24, 27].

We have previously described two single-domain frag-ments of camelid heavy-chain antibodies [31], termed

‘nanobodies’[32], denoted NbSyn2 [33] and NbSyn87 [34], which bind to distinctive epitopes within the C-terminal region of monomericɑS (residues 137–140 and 118–131, respectively). These nanobodies, which were raised against and bind to monomericɑS, also recognize fibrillar forms of ɑS in which the C-terminal region is exposed [29], allowing the use of these nanobodies as biophysical probes to study the properties of ɑS fibrils [34]. Moreover, one of these nanobodies, NbSyn87, when expressed as a genetic fusion protein with a PEST proteasomal degradation tag, was found to reduce toxicity in cell-lines overexpressing ɑS as a result of the specific degradation of monomeric ɑS, thereby significantly reducing proteostatic stress [35].

In order to understand such phenomena in molecular detail, it is important to characterize the impact of nano-body binding on the overall ɑS aggregation process in vitro, and in particular its effect on the formation of the highly toxic oligomers that are linked to cellular damage. It is known from previous studies that the C-terminal region ofɑS is exposed in at least some of its aggregated intermediate forms, such as oligomers [36], as well as monomers and fibrils, and that targeting of this region by antibodies has been recognized to have protective effects [19]. Therefore, it is plausible that the nanobodies and

other C-terminal-binding antibodies can affect the forma-tion and properties of these oligomers.

Since the pre-fibrillar oligomers are generally transient and heterogeneous, and are typically present at very low concentrations relative to ɑS monomers, they are diffi-cult to study by conventional bulk techniques. We have previously utilized single-molecule Förster resonance energy transfer (sm-FRET) measurements in order to characterize the oligomerization of ɑS in considerable detail [13, 37]. Using this technique, we identified two distinct types of ɑS oligomers that were populated sequentially prior to the formation ofɑS fibrils [13]. The two oligomer types had distinct FRET efficiency distri-butions and were therefore termed “low-FRET” and

“high-FRET” oligomers [13]. The differences in their photophysical properties, as well as their different kinet-ics of formation, stabilities towards dissociation and enzymatic proteolysis, and toxicity to cells indicated that these two oligomer forms were structurally distinct intermediates generated in the process of the formation ofɑS fibrils [13, 38]. In particular, the initial assembly of low-FRET oligomers was followed by slow conformational conversion into high-FRET oligomers prior to fibril for-mation. The accumulation of high-FRET oligomers was associated with the highest level of cytotoxicity, pointing to this specific oligomer type as the most damaging species formed duringɑS aggregation.

In this study, we set out to characterize in detail the effects of NbSyn2 and NbSyn87 on the aggregation ofɑS in vitro. First, we investigated fibril formation using a combination of biophysical experiments to monitor the kinetics of this process, and compared the properties of the various resulting fibrillar aggregates. Second, we employed sm-FRET techniques to explore how the pres-ence of NbSyn87 and NbSyn2 affects the formation of the low-FRET and high-FRET oligomers. We identi-fied the ability of the nanobodies to inhibit the for-mation of ɑS fibrils and to destabilize toxic high-FRET oligomers of ɑS, and explored how the latter process af-fects oligomer-induced cytotoxicity.


NbSyn2 and NbSyn87 inhibit the formation, maturation, and elongation ofɑS fibrils


NbSyn2, NbSyn87, or a control nanobody, cAbHuL5g (NbHul5g) [39]; the latter is a lysozyme-specific nano-body [39] and is therefore not expected to bind toɑS.

From Eq. 1 (Methods), taking the dissociation constants of NbSyn2 and NbSyn87 for monomericɑS, respectively, as approximately 264 nM and 42 nM [34] at 37 °C, we calculated that the freeɑS concentration under these con-ditions was approximately 260 nM and 40 nM, respect-ively, thereby approximating full saturation of all ɑS binding sites. The samples were incubated at physiologic-ally relevant pH and ionic strengths (as detailed in the Methods section) at 37 °C with agitation to promote fi-bril formation; the resulting kinetic profiles of fifi-bril formation are shown in Fig. 1a.

The aggregation kinetics of ɑS in the absence of nanobodies are in good agreement with our previ-ously reported bulk kinetic measurements of ɑS under similar incubation conditions [13, 37]. In contrast, moderate inhibition of fibril formation was observed in the presence of NbSyn2, whereas solutions incu-bated in the presence of NbSyn87 gave rise to much lower ThT fluorescence signals over the entire dur-ation of the experiment, indicating a stronger inhib-ition of ɑS fibril formation in the presence of this nanobody [13, 37]. In addition, we confirmed the in-hibition of the formation of ThT-active aggregates in the presence of NbSyn2 and NbSyn87 by total in-ternal reflection fluorescence microscopy imaging of the solutions extracted during the process of the ag-gregation reaction (Additional file 1: Figure S1).

Subsequently, we characterized the end products of aggregation using atomic force microscopy (AFM). In agreement with the findings from the ThT fluores-cence assays, the images obtained in this way showed the presence of large numbers of amyloid fibrils in the solutions of ɑS that had been incubated in the absence of nanobodies or in the presence of NbHul5g or NbSyn2 (Fig. 1b). In the samples of ɑS incubated with NbSyn87, only small and apparently spherical aggregates along with monomeric species were observed, and no large fibrils could be detected (Fig. 1b). Comparison of average fibril heights from AFM maps revealed that fibrils formed in the presence of NbSyn2 were thinner than fibrils formed in the absence of nanobodies or in the presence of NbHul5g (Fig. 1c and Additional file 1: Figure S1), indicating that nanobo-dies impaired the maturation of ɑS fibrils. The differ-ences in the fibril-forming capability of ɑS identified in the presence of the two ɑS-specific nanobodies might be due to the differences in the epitopes they bind. NbSyn87 binds with higher affinity and closer to the NAC region than does NbSyn2, and thereby might slow fibril formation due to steric hindrance and promote the formation of smaller fibrils (Fig. 1b).

We also performed quartz crystal microbalance experi-ments [40, 41] in order to measure the effects of NbSyn2 and NbSyn87 on the elongation rates of pre-formed ɑS fibrils upon their incubation with either monomeric ɑS, or with a stoichiometric ratio of monomeric ɑS and NbSyn87, NbSyn2, or NbHul5g (Additional file 2). It was found that the elongation rate ofɑS fibrils markedly decreased in the presence of both ɑS-specific nanobo-dies, corresponding to 30–40% of the rate in their ab-sence, as described in further detail in Additional file 2: Tables S3 and S4, and discussed in Additional file 2: Supplementary Results.

NbSyn2 and NbSyn87 impede the generation of high-FRET oligomeric species ofɑS prior to fibril formation


In sm-FRET experiments, equimolar mixtures of AF488-labeled ɑS (AF488-ɑS) and AF594-labeled ɑS (AF594-ɑS) were used at a total protein concentration of 70μM, either in the presence or absence of 140 μM of unlabeled

nanobodies. The solutions were incubated with agitation under the same conditions as in the bulk ThT assays, and aliquots were withdrawn at regular time intervals during the incubation, diluted 105-fold to generate appropriate


conditions for single-molecule detection, and immediately analyzed by sm-FRET. We analyzed the solutions both fol-lowing their dilution into aqueous buffer of the same com-position as that used for the incubations (PBS, see in Methods), and following dilution into deionized water; we have recently shown that dilution into low ionic strength solutions enables an improved separation of the low- and high-FRET oligomers [38]. We measured the average brightness of AF488-ɑS and AF594-ɑS in the presence of the unlabeled nanobodies to verify that the nanobodies did not affect the fluorescence properties of the dyes in the sm-FRET experiments (Additional file 2: Supplementary

Information). In addition, we carried out control experi-ments using samples where bothɑS and nanobodies were labeled with AF to confirm that the nanobodies were fully dissociated from ɑS during the detection step under both solution conditions; the results were consistent with previ-ously reported Kdvalues [34], as detailed in Additional file 2:

Supplementary Methods and Tables S1 and S2.

The resulting kinetic profiles ofɑS monomer depletion and oligomer formation are shown in Fig. 1d–g. The most rapid decrease in the monomer concentration, deduced from the monomer burst-rates [37], occurred in the samples containing 70 μM ɑS alone or in the


presence of the control NbHul5g (Fig. 1d, e). However, samples containing NbSyn87 and NbSyn2 showed sig-nificantly slower monomer depletion. Based on the monomer depletion data derived from sm-FRET, after 30 h of aggregation, approximately 70% of the sample was aggregated in the ɑS-only reaction, 90% in the ɑS plus control nanobody, and approximately 40% in the samples containing both ɑS and NbSyn87 or NbSyn2. The oligomers were formed in all samples, and comprised less than 2% of the samples ofɑS (Fig. 1d–g).

Transmission electron microscopy (TEM) images of the samples at incubation times longer than 100 h con-firmed the presence of abundant quantities of amyloid fibrils in the solutions containing ɑS alone or in the presence of NbHul5g and of NbSyn2, while only oligo-mers and short protofibrils were detected in the pres-ence of NbSyn87 (Additional file 3: Figure S2b). These results confirm that the nanobodies inhibit ɑS fibril for-mation, and are therefore in good agreement with the bulk ThT fluorescence data and AFM results obtained for wt unlabeledɑS as detailed above (Fig. 1b).

To elucidate the effects of the nanobodies on the formation of the low-FRET and high-FRET oligomers, individual FRET efficiency histograms of the samples re-corded following dilution into water were analyzed. These showed clear differences between the control samples and the samples containing ɑS-specific NbSyn2 and NbSyn87 (Fig. 2). As in our previous work [13, 37], we separated the FRET efficiency histograms into two groups, corresponding to the histograms derived from

“small” oligomers containing 2–5 apparent monomer units (denoted as “small-mers”) and oligomers contain-ing 6–150 apparent monomer units (“large-mers”), based on the number of peaks that were resolvable in the FRET efficiency histograms, as previously reported [37]. This procedure allows us to identify the time-dependent changes readily, which are most clearly evident in the histograms for the large oligomers formed during aggregation (Fig. 2).

For the samples ofɑS alone, andɑS in the presence of control NbHul5g, two peaks could be observed in the FRET efficiency histograms of the large-mers (Fig. 2), corresponding to the two previously identified oligomer populations [13, 37, 38]. Low-FRET oligomers are char-acterized by a population centered at an average FRET efficiency value (E) of 0.5, and high-FRET oligomers are indicated by the peak centered at E value of approxi-mately 0.8 (Fig. 2). The high-FRET population was clearly distinguishable after 24 h of aggregation and was dominant by 30 h of incubation both in the absence of nanobodies and in the presence of the control nanobody (Fig. 2), in good agreement with our previously reported kinetics of high-FRET oligomer formation [13, 37]. The mild acceleration of the formation of high-FRET species

in the presence of the control nanobody NbHul5g com-pared to theɑS-only sample might be due to the crowd-ing effect or transient interactions of NbHul5g with ɑS. Instead of exhibiting two populations at times longer than 24 h, however, the FRET histograms of oligomers formed in the presence of ɑS-specific nanobodies showed a single broad peak. The appearance of these histograms resembles those that had been obtained previously for ɑS solutions at low concentrations, in which high-FRET oligomers were not formed at high levels, yet were not fully absent [37]. Therefore, the appearance of the FRET efficiency histograms suggests that, in the presence of ɑS-specific nanobodies, the for-mation of high-FRET oligomers is impeded.

To characterize quantitatively the observed differences inɑS oligomer populations either in the absence or pres-ence of the nanobodies, we carried out a detailed kinetic analysis by fitting the experimental aggregation profiles to a nucleation-conversion-polymerization model similar to that reported previously [37]. A description of the analysis and the rate constants obtained for the individ-ual microscopic steps of the aggregation process are available in Additional file 2: Supplementary Information and Additional file 4: Figure S3. We achieved the closest agreement with experimental data by assuming that both of the ɑS-specific nanobodies accelerate the reverse microscopic reaction steps, i.e., the conformational conversion from high-FRET oligomers to low-FRET oligomers and subsequently to monomers. This increase in rate results in the net inhibition of the overall forward aggregation process in the presence of NbSyn2 and NbSyn87, and successfully explains the experimentally observed reduced proportion of high-FRET oligomers and the altered kinetics of monomer depletion and fibril formation. Furthermore, this analysis predicted the distinct accumulation of low-FRET oligomers over lon-ger aggregation times (Additional file 4: Figure S3). In the following section we discuss direct evidence for these phenomena.

NbSyn2 and NbSyn87 enhance the conversion of high-FRET into low-FRET oligomeric species


Subsequently, two molar equivalents of unlabeled NbSyn2, NbSyn87 or NbHul5g were added to these sam-ples prior to sm-FRET measurement. The representa-tive contour plots of FRET efficiencies and apparent oligomer sizes, as well as the FRET efficiency histo-grams of each sample (Fig. 3) show highly reprodu-cible results as nearly identical outcomes were observed in at least five independent experiments where fresh nanobodies were added to pre-formed high-FRET oligomers. The addition of NbSyn2 or NbSyn87 resulted in a reproducible decrease of the mean FRET efficiency values (Fig. 3b). These changes in FRET efficiencies cannot be explained by optical effects, e.g., quenching of fluorescence due to the binding of the nanobodies to the oligomeric species, because the samples were diluted by a factor of 105, bringing the concentration of the NbSyn87 and NbSyn2 significantly below the Kd values of the inter-action of the nanobodies with monomeric ɑS, as in all previous sm-FRET measurements [34]. In addition, the koff values for NbSyn87 and NbSyn2 are the order

of 0.01 and 0.1 s–1, respectively (Additional file 2: Supplementary Methods), indicating that the complexes would dissociate upon dilution, an assumption that was confirmed by the experimentally observed absence of

coincidence between the labeled nanobody andɑS under our detection conditions.

The changes in FRET efficiency values following the addition of NbSyn2 or NbSyn87 can therefore be attrib-uted solely to the nanobody-induced conformational conversion of the high-FRET oligomers to the less-ordered low-FRET oligomers, in agreement with the re-sults of the kinetic analysis. This conversion process was particularly fast in the presence of NbSyn87, with a rate constant of 1.0 ± 0.5 h–1 (Additional file 4: Figure S3); the more highly pronounced effect of NbSyn87 com-pared to NbSyn2 in this experiment is attributable to its higher affinity for ɑS. The oligomers will remain in the low-FRET structure during the analysis as the conform-ational reorganization from low-FRET to high-FRET oligomers in the absence of the nanobodies is character-ized by a high energy barrier, reflected in a half-time of several hours [13].

The conformational conversion from high- to low-FRET oligomers described here was observed upon the addition of two molar equivalents of nanobodies. We also carried out titrations to determine the lowest stoi-chiometric ratio of the nanobodies to ɑS at which this behavior is observable. The reduction in FRET efficien-cies in this experiment could be clearly observed down


to 0.5 molar equivalents of the nanobodies, while at concentrations below 0.25 molar equivalents this process was too slow to be observed even upon incubation for 10 h (Additional file 5: Figure S4).

The reduced population of high-FRET oligomers in the presence of NbSyn87 and NbSyn2 leads to significantly reduced cellular damage

The observation that NbSyn2 and NbSyn87 impair the formation of high-FRET oligomer types during the aggregation process and induce a more rapid conform-ational conversion of pre-formed high-FRET oligomers into low-FRET species, prompted us to test if these effects could result in an overall decrease in the average stability and cytotoxicity of the oligomers generated at the early stages of the aggregation process ofɑS.

It has previously been shown that high-FRET oligo-mers are more resistant towards digestion by proteinase

K than are low-FRET oligomers, an observation that serves as an additional indication of the structural differ-ences between these two oligomer types [13]. To deter-mine whether or not the populations of oligomers formed in the presence of the nanobodies were more susceptible to proteinase K digestion than these formed in their absence, we exposed ɑS samples after 29 h of incubation under aggregating conditions to varying concentrations of proteinase K. The resulting digestion profiles are shown in Fig. 4a, and representative contour plots of FRET efficiencies and size distributions de-rived for individual samples in this assay are given in Additional file 6: Figure S5. From Fig. 4a, the popula-tions of oligomeric species formed in the presence of NbSyn2 or NbSyn87 were more susceptible to proteinase K digestion than those formed in the presence of NbHul5g. This result is consistent with a lower proportion of more compact high-FRET oligomers in the solutions


containing NbSyn2 and NbSyn87 than in the presence of NbHul5g, supporting the sm-FRET findings on the rates of interconversion. Subsequently, we measured whether or not the aggregates generated during the 29 h incuba-tion period in the presence ofɑS-specific nanobodies were more degradable in comparison to the aggregates formed in the presence of the control nanobody by exposing these samples to the 26S proteasome for 24 h, and quantifying the numbers of non-degraded oligomers by sm-FRET, as described fully in Additional file 2: Supplementary Infor-mation. The results of this experiment indicate that the oligomer populations formed in the presence of the two specific nanobodies could be degraded by the proteasome to a greater extent than the aggregates formed in the presence of the control nanobody (Fig. 4b).

We then investigated if the inhibitory effect of the nanobodies at early stages of the aggregation reaction could result in a change in oligomer-induced cellular damage. High-FRET oligomers have been found in pre-vious studies to induce significantly higher levels of cellular toxicity in rat mid-brain neuronal cultures than the low-FRET species [13, 37, 42]. Aliquots were ex-tracted from samples of ɑS undergoing aggregation in the absence or presence of nanobodies after incubation for 29 h, and the production of reactive oxygen species (ROS) was measured following the application of these solutions to primary co-cultures of neurons and astrocytes (Additional file 2: Supplementary Informa-tion). The results show that the presence of NbSyn87 or NbSyn2 led to a significantly reduced rate of ROS pro-duction in comparison to the samples generated in their absence or in the presence of the control nanobody NbHul5g (Fig. 4c). This observed reduction in the oligomer-induced ROS indicates a reduced proportion of toxic high-FRET oligomers and supports the sm-FRET and proteinase K digestion results described above. We also investigated the ability of ɑS oligomer populations prepared in the presence or absence of nanobodies to in-duce cell death (see Additional file 2: Supplementary In-formation for details).

We found that the incubation of cells with ɑS aggre-gated in the presence of either NbSyn87 or NbSyn2 led to a significant reduction in cell death in comparison to the incubation of ɑS alone (Fig. 4d), indicating in the former the presence of a lower proportion of high-FRET compared to low-FRET oligomers. Apart from the significantly reduced cytotoxicity upon application of nanobody-bound oligomers in comparison to the oligo-mers of ɑS alone in ROS and cell death assays, it was found that the samples containing NbSyn2 reduced the cytotoxicity more effectively in comparison to NbSyn87 (Fig. 4c, d). While the overall reduction of cytotoxicity in comparison to theɑS-only samples is entirely consistent with the measured ability of NbSyn87 and NbSyn2 to

convert toxic high-FRET oligomers to low-FRET oligo-mers in aqueous solution, the difference between the two ɑS-specific nanobodies suggests the influence of other factors in the cell milieu in addition to the struc-ture of ɑS oligomers. The lower protective effect of NbSyn87 compared to NbSyn2 in these experiments might be due to the high positive charge of NbSyn87 (pI > 9.0) and increased interaction with the cell mem-branes, as well as due to its ability to abolish ɑS fibril formation leading to the increased oligomer load. More detailed comparison of the reduction of toxicity by NbSyn2 and NbSyn87 in the cellular environment remains to be made in future studies.

Finally, we tested whether or not the oligomers formed in the presence of the ɑS-specific nanobodies reduced the pro-inflammatory activation of microglial cells by quantifying the concentration of the released pro-inflammatory cytokine, the tumor necrosis factor alpha (TNF-ɑ) protein [43], upon their incubation with ɑS solutions after aggregation for 29 h. The experimental procedure is described in Additional file 2: Supplementary Information, and the results show that the application of ɑS solutions aggregated with NbSyn2 and NbSyn87 resulted in a significantly reduced activation of microglia in comparison to ɑS alone and/or NbHul5g-containing samples (Fig. 4e).

Taken together, the comparative assays in Fig. 4 indicate a lower average stability of the ɑS oligomer populations generated in the presence of the ɑS-specific nanobodies and a reduced propensity to cause cellular damage. These findings are completely consistent with the conclusion that the population of the more toxic high-FRET oligomer type is reduced during the aggregation process of ɑS in the presence of NbSyn87 and NbSyn2.



The ability of the nanobodies to promote the direct conformational conversion from more toxic to less toxic oligomeric species demonstrated in this study indicates the existence of an influential and unexplored mode of action of nanobodies or antibodies. Previously estab-lished modes of action of antibodies and nanobodies in vitro have included those that alter the equilibrium be-tween fibrils, oligomers, and monomers [44], which can take a finite length of time to make sufficient changes in oligomer concentrations, and those that target oligomers to decrease their binding to cell membranes [11, 45]. The rapid conformational conversion by nanobodies of toxic oligomers to less toxic oligomers identified in our experiments has a direct effect on their ability to cause cellular damage. The results suggest, therefore, that the ability of nanobodies to promote conformational conver-sion of oligomers could have important potential for the development of therapeutic strategies. Since our study has been performed in vitro and at high concentrations of ɑS, it will be of interest to elucidate the role of this mechanism under a more complex biological environ-ment in vivo in future investigations.

The ability of the nanobodies to destabilize high-FRET oligomers observed here may be due to a combination of their significantly smaller size than conventional anti-bodies [31] and their binding to the accessible C-terminal region of the protein [33, 34]. Antibodies binding to the C-terminal region of ɑS have previously been shown to decrease the production of highly aggregation-prone trun-cated forms of ɑS lacking the C-terminal region [46]. Thus, steric effects could be an important feature in enab-ling the smaller nanobodies to interact more effectively with oligomers than larger antibodies and further com-parison of the action of nanobodies and conventional anti-bodies is warranted. Optimization of this destabilization effect in future work by probing the nanobody binding site in further detail may be possible through approaches that can distinguish between different oligomer structures, and hence measure the extent of destabilization.


This study has involved the detailed biophysical characterization of the role ofɑS-specific nanobodies on the aggregation of ɑS in vitro, including the use of single-molecule fluorescence techniques. It shows that nanobodies targeting the C-terminal region inhibit the aggregation of ɑS not only by inhibiting its aggregation and elongation processes, but also by inhibiting the conformational conversion of oligomers formed prior to fibril formation. The latter effect leads to the decrease in oligomer stability and significantly reduces the cellular damage resulting from the effects of oligomeric species. Our work reveals an important mode of action of nano-bodies, namely their ability rapidly to convert already

formed toxic oligomers to less toxic ones. Such a mode of action has the potential to form the basis for a thera-peutic strategy to combat PD and related protein misfolding conditions.


Expression and purification of nanobodies andɑS

NbSyn87, NbSyn2, and NbHul5g were expressed in the periplasm ofE. colicells and purified using immobilized metal affinity chromatography and size-exclusion chro-matography as previously described [33]. The amino acid sequences of NbSyn87 and NbSyn2 were as previously reported [34]. The sequence and structure of NbHul5g and the details of the construction of its more stable variant NbHul5g has been previously reported [39]. Ex-pression and purification of wt and the alanine to cyst-eine mutant at position 90 (A90C) ɑS were carried out according to published protocols [47].

ThT aggregation assays

Solutions of 600μL of wtɑS at a concentration of 70μM either with or without 2 molar equivalents of NbSyn87, NbSyn2, or NbHul5g in PBS buffer (10 mM phosphate, pH 7.5, 100 mM NaCl, 2 mM EDTA, and 0.1% NaN3)

were incubated for a period of 107 h at 37 °C with con-stant agitation at 200 rpm (New Brunswick Scientific Innova 43). Aliquots of 5μL were removed at various time points and added to a 95μL ThT solution (20μM) in PBS buffer. ThT fluorescence was measured using a Cary Eclipse fluorimeter (Varian) with excitation and emission wavelengths of 440 and 480 nm, respectively. The mean maximum intensities from triplicate measurements were reported.

AFM measurements


performed by SPIP (Image Metrology) software. Analysis of mean fibril heights was carried out as previously reported [48] and described in detail in Additional file 2: Supplementary Information.

ɑS labeling

The A90C mutant variant of ɑS was labeled with either maleimide-modified AF488 or AF594 dyes (Life Technolo-gies) via the cysteine thiol moiety as previously reported [13, 49]. The labeled protein was purified from the excess of free dye using a P10 desalting column with Sephadex G25 matrix (GE Healthcare) and concentrated using Ami-con Ultra CentriAmi-cons (Millipore), divided into aliquots, flash frozen and stored at−80 °C. Each aliquot was thawed immediately and used only once.

Estimation of the concentration of freeɑS

The concentration of freeɑS was estimated as the differ-ence between the total starting concentration of ɑS and the concentration of boundɑS in the presence of nano-bodies, calculated according to:


½ bound¼

1 2


½ þ½Nb þKd

− ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffið½ þas ½Nb þKdÞ2−4½ as½Nb


Þ ð1Þ

where [as]bound is the concentration of bound ɑS, [as]

and [Nb] are the starting concentrations ofɑS and nano-body, andKd is the corresponding dissociation constant


Sm-FRET experiments

The experiments were carried out according to previ-ously published procedures [37, 38], utilizing a custom-built single-molecule setup as already described [50] and microfluidic devices fabricated as previously reported [38]. The single-molecule instrumentation has been previously described in detail [37]. For the aggregation reactions, equimolar concentrations of AF488- and AF594-labeled A90C ɑS (AF488-ɑS and AF594-ɑS) in PBS were combined to a final volume of 300μL, bring-ing the total ɑS concentration to 70 μM, either in the presence or absence of 140 μM of unlabeled NbSyn87, NbSyn2, or NbHul5g. At least three separate samples were prepared and analyzed at each protein combin-ation. The solutions were incubated in the dark for up to 50 h under the same incubation conditions as the samples for ThT assays (37 °C, with agitation). Aliquots were withdrawn at regular time intervals and diluted 2.5 × 105 fold by serial dilution in either PBS buffer or deionized water (Milli-Q) to give a concentration

suitable for analysis in the single molecule regime. Im-mediately upon dilution, the solution was passed at a constant rate of 2 cm s–1 (syringe pump PHD2000, Harvard Apparatus) through a channel of a microfluidic device mounted on the single-molecule confocal micro-scope (Nikon Eclipse Ti-U). The 488 nm laser beam was focused into the center of the channel (10μm along ver-tical direction). The experimental data consisted of syn-chronous time-binned fluorescence output from the donor (AF488) and the acceptor (AF594) channels, acquired for 400 s per aliquot (80 frames, 100,000 bins frame–1, 50 μs bin-width). The absence of oligomer dissociation during sm-FRET detection was previously confirmed [37], and the estimated oligomer concentra-tions were previously found to agree with the oligomer concentrations at bulk conditions [13].

Sm-FRET data analysis

The resulting sm-FRET data were analyzed as previously reported [37, 38], using custom-written Igor Pro code (Wavemetrics). Time-bins with intensities above 15 photons.bin–1 were selected in both the donor and acceptor channel (AND criterion) [51]. Simultaneous fluorescence bursts above the threshold were assigned to oligomeric events and the non-simultaneous donor bursts above the threshold correspond to monomeric ɑS. The intensities of the selected photon bursts were corrected for the crosstalk from donor to acceptor chan-nel (13%), and the autofluorescence in the acceptor channel (1.3 photons.bin–1). For each oligomeric burst, the two key parameters calculated were the apparent size of the oligomer (Size) and the FRET efficiency (E), defined as:


IAþγID ð


−1I A

Imonomer ð

where ID is the donor intensity in the presence of an

acceptor, IA is the acceptor intensity, γ is the gamma

factor, which corrects for the differences in detection efficiencies of the two fluorescent probes and their quantum yields (γ = 0.99), and Imonomer is the mean


were assumed to be due to fibrillar species and were excluded from the analysis, as reported in our previous study [38]. Subsequently, both the monomer and the oligomer bursts were converted into corresponding bulk ɑS concentrations by taking into account the dilution factor, as previously reported [37].

Additional files

Additional file 1: Figure S1. Additional results. (a) AFM images of starting monomeric solutions of wild-type (wt)ɑS prior to incubation with agi-tation. (b) Total internal reflection fluorescence microscopy results. Left: repre-sentative sum-image in the ThT emission channel (100 frames) and the corresponding reconstruction image in the NR channel (2000 frames). Right: comparison of the total numbers of aggregates and percent of ThT-active aggregates formed in the wtɑS-only and wtɑS + nanobody solutions at the same time-point of the aggregation process. (c) Scatter plots and statistical comparison of the distributions of average fibril heights derived from AFM maps (Fig. 1b, c, main text). (d) Quartz crystal microbalance recordings usingɑS (21μM), nanobody alone (21μM) or 1:1 mixture ofɑS with nanobody (21μM : 21μM) or control peptide (42μM). (TIF 4648 kb)

Additional file 2:Supplementary Information. (PDF 462 kb) Additional file 3: Figure S2. Results of control bulk ThT experiments and TEM imaging of labeledɑS. (a) Progression of fibril formation, monitored by ThT fluorescence emission from either unlabeled wild-type (black) or AF-594 labeledɑS at 70μM (n = 3, SD). (b) TEM images of aggre-gates formed in 70μM 1:1 AF488:AF594 dual-labeledɑS solutions. Top: la-beledɑS solutions at different time-points during aggregation. Bottom:ɑS solutions in the presence of 140μM of unlabeled NbHul5g, NbSyn2, and NbSyn87 after more than 100 h incubation with agitation. Large amyloid fibrils and fibrillar fragments were observed in all samples at this time, except in the presence of NbSyn87, where short protofibrils were present along with oligomeric aggregates. (TIF 4331 kb)

Additional file 4: Figure S3. Kinetic analysis ofɑS aggregation. (a) Schematic representation of the nucleation-conversion-polymerization model. MonomericɑS form low-FRET oligomers with rate constantkn and an average reaction ordernc. Low-FRET oligomers convert into high-FRET oligomers by a first-order reaction with a rate constantk1c, which is

followed by a first-order conversion to fibrils, with a rate constantk2c.

Once formed, fibrils grow by monomer addition with a length-independent rate constantke. The conversion steps between oligomer types are treated as size independent, andk1cis set to be equal tok2c.

First-order reverse conversion reactions from high-FRET to low-FRET oligo-mers and from low-FRET oligooligo-mers to monooligo-mers are introduced, with rate constants kc1


and kn˜, respectively. (b–e) This model was fitted globally to

the kinetic data ofɑS aggregation at 70μM in the absence or the presence of 140μM of nanobodies. The global fits (dashed lines) were performed with parameterskn= (1.0 ± 0.5) × 10−3h−1,k

e= 0.16

± 0.08μM−1h−1,k1c=k2c= 0.12 ± 0.04 h−1, k˜n¼kc1


0 h1,nc= 1 ± 0.1,mtot= 70μM for reactions containingɑS only andɑS with NbHul5g. The reverse reaction from high-FRET oligomers to low-FRET oligomers was found to be accelerated in the presence of NbSyn2 and NbSyn87, with k˜n¼0:20:1 h1and kc1


10:5 h1forɑS aggregation in the presence of NbSyn2, and with k˜n¼0:250:1 h1

and kc1


105 h1forɑS aggregation in the presence of NbSyn87, with all remaining parameters unchanged. (f) Predictions of the concentrations of low-FRET and high-FRET oligomers during 100 h ofɑS aggregation in the presence or absence of nanobodies. (TIF 710 kb)

Additional file 5: Figure S4. Representative FRET efficiency histograms from the‘reverse’sm-FRET experiments, analogous to those shown in Fig. 3 (main text). (a) Pre-formed high-FRET oligomers were formed in a forward incubation of monomericɑS (70μM in PBS, 29 h, shaking at 37 °C). To the pre-formed oligomer solutions, either 1, 0.5, or 0.25 molar equivalents of nanobodies, NbSyn87 (b) or NbSyn2 (c), were added and sm-FRET detec-tion was carried out within 5 min after the addidetec-tion. In the case of 0.25

equivalents, the samples were further incubated at 37 °C under quies-cent conditions (in low-binding test-tubes), and sm-FRET analysis repeated. (TIF 877 kb)

Additional file 6: Figure S5. Representative contour plots of FRET efficiency and size after proteinase K digestion of 29-h time-points by differ-ent concdiffer-entrations of proteinase K (Fig. 4a, main text). (a) Control sample containing NbHul5g is less degradable in comparison to the samples pre-pared in the presence of NbSyn2 (b) and NbSyn87 (c), as indicated by the presence of higher oligomer fraction remaining in the sample upon incuba-tion with proteinase K. This is consistent with the presence of high-FRET olig-omers in the sample. (TIF 1028 kb)

Additional file 7:Supporting Data Values. (XLSX 27 kb)

Additional file 8: Figure S6. Representative result from the reactive oxygen species measurements presented in main text, Fig. 4c. Application of 500 nM of AF-labeledɑS solution induced an increase in the ratio of dihydroethydium (HEt) fluorescence between its oxidized and non-oxidized forms. The time whenɑS was applied is marked with the grey bracket on the plot. A higher increase in HEt ratio is observed upon application ofɑS solutions containing control NbHul5g, suggesting that oligomers formed in its presence are more damaging in comparison to the oligomers formed in the presence ofɑS-specific nanobodies. (TIF 108 kb)


We are grateful to Ewa Klimont and Swapan Preet for the expression and purification of A90CɑS, and thank Dr. Yu Ye for the reagents used in the proteasome degradation assays.


MI was funded by a Dr Tayyeb Hussain Scholarship and the ERC (669237). LH was funded by a China Scholarship Council and NSFC 11204150. TG was a recipient of a studentship from Parkinsons UK (H-0903). EDG was supported by the Medical Research Council (MRC G1002272). DK was funded by the ERC (669237) and the Royal Society, CMD, TPJK and MV by the Wellcome Trust.

Availability of data and materials

All experimental methods and results generated during this study are included in the article and in Additional file 2: Supplementary Information (SI.pdf), available online. The individual data values for the datasets in Figs. 1 and 4 are included as an Additional file 7: Supporting Data Values.xls.


MI designed and performed the single-molecule fluorescence experiments and data analysis. EDG performed the nanobody preparation, bulk thioflavin T assays, and aided with single-molecule experiments. LH performed the kin-etic analysis. MHH aided with the single-molecule experiments and the de-sign of the project. MHL, MLC, and CDH performed the cell experiments. SG and CEB designed the cell experiments. FSR performed the AFM measure-ments and analysis. AKB performed the quartz crystal microbalance experi-ments. TG performed the nanobody preparation. JEL and SFL performed total internal reflection fluorescence microscopy imaging and data analysis. TPJK and MV supervised the study. CMD, EDG, and DK designed and super-vised the study. MI, EDG, DK, and CMD wrote the paper, with contributions from all authors. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing financial interests.

Consent for publication Not applicable.

Ethics approval and consent to participate Not applicable.

Publisher’s Note


Author details

1Department of Chemistry, University of Cambridge, Lensfield Road,

Cambridge CB2 1EW, UK.2Zhou Pei-Yuan Center for Applied Mathematics,

Tsinghua University, Beijing 100084, China.3Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR, UK.4Department of Molecular Neuroscience, University College

London, Institute of Neurology, Queen Square, London WC1N 3BG, UK.


Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.6Present address: Healx Ltd., St Johns

Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK.7Present address:

Astra Zeneca, Innovative Medicines Discovery Sciences Unit 310, Darwin Building, Cambridge Science Park, Milton Road, Cambridge CB4 0WG, UK.

8Present address: Institute of Physical Biology, University of Düsseldorf,

Universitätsstr. 1, 40225 Düsseldorf, Germany.

Received: 11 April 2017 Accepted: 6 June 2017


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Fig. 1 Aggregation kinetics of 70 μM ɑS in the absence and presence of nanobodies. a Bulk kinetic profiles of fibril formation, monitored by ThTfluorescence (n = 3, mean ± SEM)
Fig. 2 Representative FRET efficiency histograms obtained for different time points during the first 30 h of aggregation, recorded upon dilution intodominant and corresponds to high-FRET oligomers
Fig. 3 Results of sm-FRET measurements after addition of two equivalents of unlabeled nanobodies to pre-formed high-FRET oligomers
Fig. 4 Comparative assays showing the relative stability and cytotoxicity of0.001); andversus time.the absence oferslips,after the application of 29 h timepoints ofequivalents of NbHul5g (n = 3, SEM, ɑS oligomers generated after aggregation for 29 h in the


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